This application is responsive to the objective of the RFA which calls for development of novel technologies to extend the duration of antigen-specific tolerance; and development or application of cell and tissue engineering methods to predictably induce tolerance rather than immunity. The goal is to develop a "chemical genetics" approach to T cell anergy. Libraries of small organic molecules will be screened at the Institute for Chemistry and Cell Biology at Harvard Medical School, with the objective of identifying compounds that induce or inhibit T cell anergy by targeting the transcription factor NFAT and the E3 ubiquitin ligase Itch, respectively. Aim 1 is to identify compounds which inhibit the interaction of NFAT with its nuclear partner AP-1 (Fos-Jun). Since NFAT without AP-1 induces T cell anergy, whereas NFAT with AP-1 is required for normal immune responses characterised by transcription of cytokine and chemokine genes, inhibitors of the NFAT:AP-1 interaction are predicted to attenuate ongoing immune responses while at the same time inducing T cell anergy. If appropriately developed for in vivo use, such inhibitors would potentially be useful in combating transplant rejection and autoimmune/atopic disease. Aim 2 is to identify inhibitors of Itch, a HECT-family E3 ligase with intrinsic ubiquitin ligase activity. Itch and two other E3 ligases, Cbl-b and Grail, are upregulated at the mRNA and protein levels in anergic T cells, where they participate in destroying key signalling molecules that are essential for T cell activation. Mice deficient in Itch or Cbl-b show a strong propensity for autoimmune disease, and their T cells cannot be anergised efficiently in vitro. Inhibitors of these E3 ligases would be predicted to interfere with T cell tolerance induction; if appropriately developed for in vivo use, they would be clinically useful in counteracting T cell tolerance to tumours. The project is innovative as required by the RFA, since it applies chemical genetics to manipulate (induce or inhibit) T Cell tolerance with cell-permeant small molecules which interfere with the functions of specific molecules known to be in the transcriptional and signalling arms, respectively, of a pathway leading to T cell anergy. The hope is that the 'information obtained from this pilot R21 proposal will eventually be extended to whole animal models of transplant and tumour rejection, allergy and autoimmune disease.